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Formyltetrahydrofolate hydrolase from Escherichia coli
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[251
[25] F o r m y l t e t r a h y d r o f o l a t e H y d r o l a s e f r o m E s c h e r i c h i a coli By
HOWARD ZALKIN
Formyltetrahydrofolate hydrolase, encoded by the Escherichia colipurU gene, is a regulatory enzyme that catalyzes the hydrolysis of Nl°-formyltetrahydrofolate (formyl-FH4)l: Formyl-FH4 +
H20
~
FH4 + formate
Formyl-FH4 hydrolase has two important roles in E. coli. First, the enzyme monitors the relative pools of FH4 and FH4 one-carbon adducts (C1-FH4) and functions to replenish FH4 when other routes of FH4 regeneration are shut down due to growth conditions that repress biosynthetic pathways for histidine, methionine, and purine nucleotides. Tetrahydrofolate is needed by serine hydroxymethyltransferase for glycine synthesis. A second role for the enzyme is to generate formate during aerobic growth. Formate is utilized by 5'-phosphoribosylglycinamide (GAR) transformylase-T for synthesis of 5'-phosphoribosyl-N-formylglycinamide (FGAR)2: GAR + formate + ATP ~ FGAR + ADP + Pi Although E. coli contains a second GAR transformylase-N that utilizes formyl-FH4 as the one-carbon donor for synthesis of FGAR, formate can contribute up to 50% of the carbon for position 8 of the purine ring in wild-type cells. 3 Mutant analysis has indicated that formyl-FH4 hydrolase supplies the formate that is utilized by the GAR transformylase-T reaction during aerobic growth. 4
Assay Method A polyglutamate derivative of formyl-FH4 is assumed to be the natural substrate for formyl-FH4 hydrolase. Although formyl-FH4 serves as a substrate, difficulty of preparation and oxygen lability limit its routine use. 1 p. L. Nagy, A. Marolewski, S. J. Benkovic, and H. Zalkin, J. Bacteriol. 177, 1292 (1995). 2 A. Marolewski, J. M. Smith, and S. J. Benkovic, Biochemistry 33, 2531 (1994). 3 I. K. D e v and R. J. Harvey, J. Biol. Chem. 257, 1980 (1982). 4 p. L. Nagy, G. M. McCorkle, and H. Zalkin, J. Bacteriol. 175, 7066 (1993).
METHODS IN ENZYMOLOGY,VOL. 281
Copyright © 1997by AcademicPress All rights of reproduction in any form reserved. 0076-6879/97 $25
[25]
FORMYLTETRAHYDROFOLATE HYDROLASE
215
10-Formyl-5,8-dideazafolate (fDDF), 5 which is not sensitive to aerobic oxidation, is the preferred substrate for routine enzyme assays. 10-Formyl5,8-dideazafolate is presently available from J. B. Hynes (Department of Pharmaceutical Chemistry, Medical University of South Carolina, Charleston, SC). An assay mixture of 100/~1 contains 50 mM Tris-HC1 (pH 7.5), 60/~M fDDF, 2 mM methionine, and enzyme. The reaction is carried out at room temperature (-23 °) and is started by the addition of enzyme. The initial rate of hydrolysis is recorded at 295 nm (Ae of 18.9 mM 1 cm 1), Enzyme Overproduction Escherichia coli p u r U has been placed under the control of the phage T7 promoter thl0 in plasmid pT7-7 to yield plasmid pT7-PU1.1 The enzyme is overproduced in E. coli BL21(DE3). A single colony of BL21(DE3)/ pT7-PU1 is used to inoculate 10 ml of LB medium (10 g of tryptone, 5 g of yeast extract, 10 g of NaC1 dissolved in 1 liter and adjusted to pH 7.0 with NaOH) supplemented with 100/zg of ampicillin per milliliter. After overnight growth six 2-liter flasks, each containing 0.5 liter of LB medium with ampicillin, are inoculated with 1 ml of the overnight culture and grown with shaking at 37° to a turbidity of 180 as measured with a Klett colorimeter using a 660-nm filter (equivalent to 0.84 optical density units at 600 nm as measured in a spectrophotometer). At this point lactose is added to a final concentration of 1% for induction of T7 polymerase and the flasks are incubated at 30° with shaking for 24 hr. Cells are harvested by centrifugation and stored frozen at - 2 0 °. Approximately 30-40% of the soluble protein is formyl-FH4 hydrolase.
Enzyme Purification The enzyme is purified approximately threefold to electrophoretic homogeneity in four steps. 1 A typical purification is summarized in Table I. After ammonium sulfate precipitation the enzyme is approximately 85-90% pure, on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The residual contaminating proteins can be removed, if required, by ion-exchange chromatography to give an essentially homogeneous enzyme. Step 1. Crude Extract. All steps are carried out at 4° in a buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, plus other additions specifically noted. Frozen cells are suspended in buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) in the ratio of 1 g of cells per 4 ml of buffer. The 5j. B. Hynes,D. E. Eason,C. M. Garrett, and P. L. Colvin,Jr., J. Med. Chem. 20, 588 (1977).
216
FOLICACID
[251
TABLE I SUMMARY OF PURIFICATION
Fraction
Volume (ml)
Protein (rag)
Disrupted cells Streptomycinsulfate Ammoniumsulfate DEAE-Sepharose
135 135 10 22
2,160 850 280 205
Specific activity (nmol/min/mg) 30 55 72 85
Total activity (nmol/min) 65,800 46,800 20,200 17,400
cell suspension is broken by two passages through a French press at 20,000 lb/in 2. Step 2. Precipitation with Streptomycin Sulfate. To the broken cell suspension, 0.1 vol of 10% (w/v) streptomycin sulfate is added slowly with stirring. After the last addition, stirring is continued for 15 min, followed by centrifugation at 18,000 g for 30 min at 4 °. The supernatant containing formyl-FH4 hydrolase is used for the next step. Step 3. Precipitation with Ammonium Sulfate. Ammonium sulfate is added slowly to 30% saturation (0.176 g/ml) with stirring. Stirring is continued for 15 min following the last addition, after which the enzyme is collected by centrifugation for 30 min at 18,000 g at 4 °. The pellet is dissolved in buffer and dialyzed overnight against 100 vol of the same buffer. Any insoluble protein is removed by centrifugation at 18,000 g for 30 min. Step 4. Chromatography on DEAE-Sepharose. A 1.5 × 5 cm column of DEAE-Sepharose is used for 200-400 mg of protein. The dialyzed enzyme is loaded onto the column, the column washed with 50 ml of buffer, and the enzyme is eluted with a 300-ml linear gradient of 0 to 0.5 M NaCI in the buffer. Fractions in the main protein peak can be checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and those fractions lacking contaminating proteins are pooled. Ordinarily, the entire protein peak can be pooled without prior analysis. The enzyme is concentrated by precipitation with ammonium sulfate, dissolved in buffer, dialyzed, and adjusted to approximately 10 mg/ml. The enzyme is stable to storage at -20 °. Enzyme Properties Formyltetrahydrofolate hydrolase i s a hexamer of 32-kDa subunits. 1 Enzyme activity is stimulated 25-fold by methionine (Ka 200 #M) and is inhibited uncompetitively by glycine. The inhibition constant for glycine is dependent on the methionine concentration and varies between less than
[25]
FORMYLTETRAHYDROFOLATE HYDROLASE
217
20/~M in the absence of methionine to 40/zM with saturating methionine. Cooperativity is observed for activation by methionine and inhibition by glycine, with Hill coefficients of 1.3 to 2.7 for methionine and 0.83 to 1.7 for glycine. The Hill coefficient for activator and inhibitor is maximal in the presence of a saturating concentration of the opposing ligand. These properties are consistent with regulatory roles for methionine and glycine. The native protein of 280 amino acids appears to be composed of three domains. A C-terminal segment, residues 85-280, is identical in 53 positions with the two domains of E. coli G A R transformylase-N. 4 5'-Phosphoribosylglycinamide transformylase-N catalyzes the reaction G A R + formylF H 4 ---> F G A R + F H 4 . 6 Formyltetrahydrofolate hydrolase corresponds to an N-terminal 84-amino acid domain fused to the 196-residue G A R transformylase-like domains. 4 Comparison of conserved residues in the two sequences and the X-ray structure of G A R transformylase 7 suggest that a binding site for formyl-FH4 but not for G A R has been retained in the C domains of the enzyme. According to this model the 84-amino acid N domain would thus function to bind methionine and glycine and regulate catalysis by the transformylase-related C domains. The kca~ value with formyl-FH4 is fourfold higher than with fDDF. However, as a result of a sevenfold lower Km for fDDF the kcat/Krn is nearly twofold higher using the substrate analog compared to formyl-FH4. Role of Enzyme One role of the enzyme is to balance pools of FH4 and C1-FH4 to ensure that synthesis of glycine can be maintained when ceils have excess purines, methionine, and histidine and biosynthetic pathways for these molecules are shut down. Purine biosynthesis regenerates FH4 from formyl-FH4 in two transformylation reactions. Methionine biosynthesis regenerates FH4 as a product of the methionine synthase reactions and the histidine biosynthetic pathway produces 5'-phosphoribosyl-4-carboxamide-5-aminoimidazole (AICAR), which is transformylated in a reaction yielding FH4. In a purU mutant lacking formyl-FH4 hydrolase, repression of purine synthesis by adenine and repression of either the methionine or histidine pathways lead to a growth requirement for glycine as a result of a deficiency in regenerating FH4 from formyI-FH4 and methyl-FH4.4 The methionine/glycine ratio thus reflects the relative pools of C]-FH4 and FH4 and regulates 6 j. Inglese, D. L. Johnson, A. Shiau, J. M. Smith, and S. J. Benkovic, Biochem~try 29, 1436 (1990). 7 R. J. Almassy, C. A. Janson, C. C. Kan, and Z. Hostomska, Proc. NaK Acad Sci. U.~A. 89, 6114 (1992).
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the hydrolase to generate FH4 or to preserve formyl-FH4 as required. 1 A high ratio of methionine to glycine reflects a high C1-FHa/FH4 ratio and is a signal to activate formyl-FH4 hydrolase to regenerate FH4 for glycine synthesis. A second role of the hydrolase is to generate formate for use by G A R transformylase-T for purine synthesis. A purN mutant lacking G A R transformylase-N requires formyl-FH4 hydrolase or an external source of formate for aerobic growth. 4 In animals formyl-FH4 dehydrogenase may function to regenerate FH4.8 Tetrahydrofolate dehydrogenase is a bifunctional enzyme that catalyzes the reaction formyl-FH4 + NADP + ~ FH4 + C O 2 q- N A D P H + H + as well as the NADP+-independent hydrolysis of formyl-FH4 to FH4 and formate. 9 An N-terminal 200-amino acid domain of the dehydrogenase is related in sequence to E. coli G A R transformylase-N but does not contain the putative regulatory domain corresponding to residues 1-84 of the E. coli formyl-FH4 hydrolase and is not regulated by methionine or glycine in response to fluctuations in FH4 and C1-FH4 pools. Acknowledgment The author's research was supported by Public Health Service Grant GM24658 from the National Institutes of Health. 8 H. Min, B. Shane, and E. L. R. Stokstad, Biochim. Biophys. Acta 967, 348 (1988). 9 R. J. Cook, R. S. Lloyd, and C. Wagner, J. Biol. Chem. 266, 4965 (1991).
[26] U s e o f 13C N u c l e a r M a g n e t i c R e s o n a n c e t o E v a l u a t e Metabolic Flux through Folate One-Carbon Pools in
S a c c h a r o m y c e s cerevisiae By
D E A N R. APPLING, EVDOKIA KASTANOS, L A U R A B. PASTERNACK,
and YAKOV Y. WOLDMAN Introduction Figure i summarizes the major pathways of folate-mediated one-carbon metabolism. In most organisms, the major source of one-carbon units is carbon 3 of serine, derived from glycolytic intermediates. The one-carbon unit is transferred to tetrahydrofolate (THF) in a reaction catalyzed by serine hydroxymethyltransferase (reaction 4, Fig. 1), generating methylene-
METHODS IN ENZYMOLOGY, VOL 281
Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. 0076-6879/97 $25
FOLICACID
[251
[25] F o r m y l t e t r a h y d r o f o l a t e H y d r o l a s e f r o m E s c h e r i c h i a coli By
HOWARD ZALKIN
Formyltetrahydrofolate hydrolase, encoded by the Escherichia colipurU gene, is a regulatory enzyme that catalyzes the hydrolysis of Nl°-formyltetrahydrofolate (formyl-FH4)l: Formyl-FH4 +
H20
~
FH4 + formate
Formyl-FH4 hydrolase has two important roles in E. coli. First, the enzyme monitors the relative pools of FH4 and FH4 one-carbon adducts (C1-FH4) and functions to replenish FH4 when other routes of FH4 regeneration are shut down due to growth conditions that repress biosynthetic pathways for histidine, methionine, and purine nucleotides. Tetrahydrofolate is needed by serine hydroxymethyltransferase for glycine synthesis. A second role for the enzyme is to generate formate during aerobic growth. Formate is utilized by 5'-phosphoribosylglycinamide (GAR) transformylase-T for synthesis of 5'-phosphoribosyl-N-formylglycinamide (FGAR)2: GAR + formate + ATP ~ FGAR + ADP + Pi Although E. coli contains a second GAR transformylase-N that utilizes formyl-FH4 as the one-carbon donor for synthesis of FGAR, formate can contribute up to 50% of the carbon for position 8 of the purine ring in wild-type cells. 3 Mutant analysis has indicated that formyl-FH4 hydrolase supplies the formate that is utilized by the GAR transformylase-T reaction during aerobic growth. 4
Assay Method A polyglutamate derivative of formyl-FH4 is assumed to be the natural substrate for formyl-FH4 hydrolase. Although formyl-FH4 serves as a substrate, difficulty of preparation and oxygen lability limit its routine use. 1 p. L. Nagy, A. Marolewski, S. J. Benkovic, and H. Zalkin, J. Bacteriol. 177, 1292 (1995). 2 A. Marolewski, J. M. Smith, and S. J. Benkovic, Biochemistry 33, 2531 (1994). 3 I. K. D e v and R. J. Harvey, J. Biol. Chem. 257, 1980 (1982). 4 p. L. Nagy, G. M. McCorkle, and H. Zalkin, J. Bacteriol. 175, 7066 (1993).
METHODS IN ENZYMOLOGY,VOL. 281
Copyright © 1997by AcademicPress All rights of reproduction in any form reserved. 0076-6879/97 $25
[25]
FORMYLTETRAHYDROFOLATE HYDROLASE
215
10-Formyl-5,8-dideazafolate (fDDF), 5 which is not sensitive to aerobic oxidation, is the preferred substrate for routine enzyme assays. 10-Formyl5,8-dideazafolate is presently available from J. B. Hynes (Department of Pharmaceutical Chemistry, Medical University of South Carolina, Charleston, SC). An assay mixture of 100/~1 contains 50 mM Tris-HC1 (pH 7.5), 60/~M fDDF, 2 mM methionine, and enzyme. The reaction is carried out at room temperature (-23 °) and is started by the addition of enzyme. The initial rate of hydrolysis is recorded at 295 nm (Ae of 18.9 mM 1 cm 1), Enzyme Overproduction Escherichia coli p u r U has been placed under the control of the phage T7 promoter thl0 in plasmid pT7-7 to yield plasmid pT7-PU1.1 The enzyme is overproduced in E. coli BL21(DE3). A single colony of BL21(DE3)/ pT7-PU1 is used to inoculate 10 ml of LB medium (10 g of tryptone, 5 g of yeast extract, 10 g of NaC1 dissolved in 1 liter and adjusted to pH 7.0 with NaOH) supplemented with 100/zg of ampicillin per milliliter. After overnight growth six 2-liter flasks, each containing 0.5 liter of LB medium with ampicillin, are inoculated with 1 ml of the overnight culture and grown with shaking at 37° to a turbidity of 180 as measured with a Klett colorimeter using a 660-nm filter (equivalent to 0.84 optical density units at 600 nm as measured in a spectrophotometer). At this point lactose is added to a final concentration of 1% for induction of T7 polymerase and the flasks are incubated at 30° with shaking for 24 hr. Cells are harvested by centrifugation and stored frozen at - 2 0 °. Approximately 30-40% of the soluble protein is formyl-FH4 hydrolase.
Enzyme Purification The enzyme is purified approximately threefold to electrophoretic homogeneity in four steps. 1 A typical purification is summarized in Table I. After ammonium sulfate precipitation the enzyme is approximately 85-90% pure, on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The residual contaminating proteins can be removed, if required, by ion-exchange chromatography to give an essentially homogeneous enzyme. Step 1. Crude Extract. All steps are carried out at 4° in a buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, plus other additions specifically noted. Frozen cells are suspended in buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) in the ratio of 1 g of cells per 4 ml of buffer. The 5j. B. Hynes,D. E. Eason,C. M. Garrett, and P. L. Colvin,Jr., J. Med. Chem. 20, 588 (1977).
216
FOLICACID
[251
TABLE I SUMMARY OF PURIFICATION
Fraction
Volume (ml)
Protein (rag)
Disrupted cells Streptomycinsulfate Ammoniumsulfate DEAE-Sepharose
135 135 10 22
2,160 850 280 205
Specific activity (nmol/min/mg) 30 55 72 85
Total activity (nmol/min) 65,800 46,800 20,200 17,400
cell suspension is broken by two passages through a French press at 20,000 lb/in 2. Step 2. Precipitation with Streptomycin Sulfate. To the broken cell suspension, 0.1 vol of 10% (w/v) streptomycin sulfate is added slowly with stirring. After the last addition, stirring is continued for 15 min, followed by centrifugation at 18,000 g for 30 min at 4 °. The supernatant containing formyl-FH4 hydrolase is used for the next step. Step 3. Precipitation with Ammonium Sulfate. Ammonium sulfate is added slowly to 30% saturation (0.176 g/ml) with stirring. Stirring is continued for 15 min following the last addition, after which the enzyme is collected by centrifugation for 30 min at 18,000 g at 4 °. The pellet is dissolved in buffer and dialyzed overnight against 100 vol of the same buffer. Any insoluble protein is removed by centrifugation at 18,000 g for 30 min. Step 4. Chromatography on DEAE-Sepharose. A 1.5 × 5 cm column of DEAE-Sepharose is used for 200-400 mg of protein. The dialyzed enzyme is loaded onto the column, the column washed with 50 ml of buffer, and the enzyme is eluted with a 300-ml linear gradient of 0 to 0.5 M NaCI in the buffer. Fractions in the main protein peak can be checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and those fractions lacking contaminating proteins are pooled. Ordinarily, the entire protein peak can be pooled without prior analysis. The enzyme is concentrated by precipitation with ammonium sulfate, dissolved in buffer, dialyzed, and adjusted to approximately 10 mg/ml. The enzyme is stable to storage at -20 °. Enzyme Properties Formyltetrahydrofolate hydrolase i s a hexamer of 32-kDa subunits. 1 Enzyme activity is stimulated 25-fold by methionine (Ka 200 #M) and is inhibited uncompetitively by glycine. The inhibition constant for glycine is dependent on the methionine concentration and varies between less than
[25]
FORMYLTETRAHYDROFOLATE HYDROLASE
217
20/~M in the absence of methionine to 40/zM with saturating methionine. Cooperativity is observed for activation by methionine and inhibition by glycine, with Hill coefficients of 1.3 to 2.7 for methionine and 0.83 to 1.7 for glycine. The Hill coefficient for activator and inhibitor is maximal in the presence of a saturating concentration of the opposing ligand. These properties are consistent with regulatory roles for methionine and glycine. The native protein of 280 amino acids appears to be composed of three domains. A C-terminal segment, residues 85-280, is identical in 53 positions with the two domains of E. coli G A R transformylase-N. 4 5'-Phosphoribosylglycinamide transformylase-N catalyzes the reaction G A R + formylF H 4 ---> F G A R + F H 4 . 6 Formyltetrahydrofolate hydrolase corresponds to an N-terminal 84-amino acid domain fused to the 196-residue G A R transformylase-like domains. 4 Comparison of conserved residues in the two sequences and the X-ray structure of G A R transformylase 7 suggest that a binding site for formyl-FH4 but not for G A R has been retained in the C domains of the enzyme. According to this model the 84-amino acid N domain would thus function to bind methionine and glycine and regulate catalysis by the transformylase-related C domains. The kca~ value with formyl-FH4 is fourfold higher than with fDDF. However, as a result of a sevenfold lower Km for fDDF the kcat/Krn is nearly twofold higher using the substrate analog compared to formyl-FH4. Role of Enzyme One role of the enzyme is to balance pools of FH4 and C1-FH4 to ensure that synthesis of glycine can be maintained when ceils have excess purines, methionine, and histidine and biosynthetic pathways for these molecules are shut down. Purine biosynthesis regenerates FH4 from formyl-FH4 in two transformylation reactions. Methionine biosynthesis regenerates FH4 as a product of the methionine synthase reactions and the histidine biosynthetic pathway produces 5'-phosphoribosyl-4-carboxamide-5-aminoimidazole (AICAR), which is transformylated in a reaction yielding FH4. In a purU mutant lacking formyl-FH4 hydrolase, repression of purine synthesis by adenine and repression of either the methionine or histidine pathways lead to a growth requirement for glycine as a result of a deficiency in regenerating FH4 from formyI-FH4 and methyl-FH4.4 The methionine/glycine ratio thus reflects the relative pools of C]-FH4 and FH4 and regulates 6 j. Inglese, D. L. Johnson, A. Shiau, J. M. Smith, and S. J. Benkovic, Biochem~try 29, 1436 (1990). 7 R. J. Almassy, C. A. Janson, C. C. Kan, and Z. Hostomska, Proc. NaK Acad Sci. U.~A. 89, 6114 (1992).
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[261
the hydrolase to generate FH4 or to preserve formyl-FH4 as required. 1 A high ratio of methionine to glycine reflects a high C1-FHa/FH4 ratio and is a signal to activate formyl-FH4 hydrolase to regenerate FH4 for glycine synthesis. A second role of the hydrolase is to generate formate for use by G A R transformylase-T for purine synthesis. A purN mutant lacking G A R transformylase-N requires formyl-FH4 hydrolase or an external source of formate for aerobic growth. 4 In animals formyl-FH4 dehydrogenase may function to regenerate FH4.8 Tetrahydrofolate dehydrogenase is a bifunctional enzyme that catalyzes the reaction formyl-FH4 + NADP + ~ FH4 + C O 2 q- N A D P H + H + as well as the NADP+-independent hydrolysis of formyl-FH4 to FH4 and formate. 9 An N-terminal 200-amino acid domain of the dehydrogenase is related in sequence to E. coli G A R transformylase-N but does not contain the putative regulatory domain corresponding to residues 1-84 of the E. coli formyl-FH4 hydrolase and is not regulated by methionine or glycine in response to fluctuations in FH4 and C1-FH4 pools. Acknowledgment The author's research was supported by Public Health Service Grant GM24658 from the National Institutes of Health. 8 H. Min, B. Shane, and E. L. R. Stokstad, Biochim. Biophys. Acta 967, 348 (1988). 9 R. J. Cook, R. S. Lloyd, and C. Wagner, J. Biol. Chem. 266, 4965 (1991).
[26] U s e o f 13C N u c l e a r M a g n e t i c R e s o n a n c e t o E v a l u a t e Metabolic Flux through Folate One-Carbon Pools in
S a c c h a r o m y c e s cerevisiae By
D E A N R. APPLING, EVDOKIA KASTANOS, L A U R A B. PASTERNACK,
and YAKOV Y. WOLDMAN Introduction Figure i summarizes the major pathways of folate-mediated one-carbon metabolism. In most organisms, the major source of one-carbon units is carbon 3 of serine, derived from glycolytic intermediates. The one-carbon unit is transferred to tetrahydrofolate (THF) in a reaction catalyzed by serine hydroxymethyltransferase (reaction 4, Fig. 1), generating methylene-
METHODS IN ENZYMOLOGY, VOL 281
Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. 0076-6879/97 $25